Generation of Dendritic Cells from Fresh and Frozen Cord Blood CD34Cells

Dendritic cells (DCs) are professional antigen-presenting cells that are required for the initiation of the immune response. DCs have been shown to be generated from CD34⁺pluripotent hematopoietic progenitor cells in the bone marrow and cord blood (CB), but relatively little is known about the effect of cryopreservation on functional maturation of DCs from hematopoietic stem cells. In this work we report the generation of DCs from cryopreserved CB CD34⁺cells. CB CD34⁺cells were cryopreserved at −80°C for 2 days. Cryopreserved CB CD34⁺cells as well as freshly isolated CB CD34⁺cells cultured with granulocyte—macrophage colony-stimulating factor (GM-CSF)/stem cell factor (SCF)/tumor necrosis factor-α (TNF-α) for 14 days gave rise to CD1a⁺/CD4⁺/CD11c⁺/CD14⁻/CD40⁺/CD80⁺/CD83⁺/CD86⁺/HLA-DR⁺cells with dendritic morphology. DCs derived from cryopreserved CB CD34⁺cells showed a similar endocytic capacity for fluorescein isothiocyanate-labeled dextran and lucifer yellow when compared with DCs derived from freshly isolated CB CD34⁺cells. Flow cytometric analysis revealed that two CC chemokine receptors (CCRs), CCR-1 and CCR-3, were expressed on the cell surface of DCs derived from both cryopreserved and freshly isolated CB CD34⁺cells, and these DCs exhibited similar chemotactic migratory capacities in response to regulated on activation normal T-cell expressed and secreted. DCs derived from cryopreserved as well as freshly isolated CB CD34⁺cells were more efficient than peripheral blood mononuclear cells in the primary allogeneic T-cell response. These results indicate that frozen CB CD34⁺cells cultured with GM-CSF/TNF-α/SCF gave rise to dendritic cells which were morphologically, phenotypically and functionally similar to DCs derived from fresh CB CD34⁺cells.     

Altered frequency of CD8+CD11c+ T cells and expression of immunosuppressive molecules in lymphoid organs of mouse model of colorectal cancer

CD11c is a member of the β2-integrin family typically used to define myeloid dendritic cells (DCs). Recent reports identify CD11c-expressing CD8⁺ T cells as a new subset of CD8⁺ regulatory T cells (Treg). Evidence exists that CD11c⁺CD8⁺ T cells may exert their effector or regulatory functions under different conditions. To date, no studies have addressed the frequency of CD11c⁺ T cells in cancer. Limited evidence exists in terms of expression of immune-checkpoint receptors, programmed cell death protein 1 (PD-1) and T-lymphocyte-associated antigen 4 (CTLA-4), as well as forkhead box P3 (Foxp3) in mouse lymphoid organs. Here, we have assessed CD11c⁺CD8⁺ and CD11c⁺CD4⁺ T cells, Foxp3, PD-1, and CTLA-4 expressing CD4⁺ T cells and CD8⁺ T cells in different tissues from three groups of male BALB/c mice—young, mature, and those with colorectal cancer (CRC). Analysis of CD3⁺CD11c⁺ T cells in the bone marrow (BM), spleen, and lymph nodes (LN) in each group showed a higher percentage of CD3⁺CD11c⁺ T cells in the BM from all groups and in the lymphoid organs of the cancer group compared with the young and mature groups. CD4^low and CD4^high cell fractions in mice BM have different expression patterns for Foxp3 and CTLA-4. We have observed a higher frequency of CD8⁺PD-1⁺ T cells in the BM, spleen, and LN of CRC mice compared with normal mice. T-cell exhaustion is associated with inhibitory receptor PD-1. According to the regulatory roles of CD11c expression in CD8⁺ T cells, we have proposed that the elevated percentage of CD11c, Foxp3, CTLA-4, and PD-1 expressing T cells were associated with immune response dysregulation in CRC.     

Stimulation of immune suppressive CD34+ cells from normal bone marrow by Lewis lung carcinoma tumors

 Progressive growth of metastatic Lewis lung carcinoma (LLC-LN7) tumors is associated with increased levels of bone-marrow-derived CD34⁺ cells having natural suppressor (NS) activity toward T cells. The present studies determined whether tumor-derived products are responsible for this induction of NS activity. Culturing normal bone marrow cells with LLC-LN7-conditioned medium (LLC-CM) or with recombinant granulocyte/macrophage-colony-stimulating factor (GM-CSF) resulted in the appearance of NS activity. The development of NS activity coincided with a prominent increase in the levels of CD34⁺ cells. That the CD34⁺ cells were responsible for the NS activity of the bone marrow cultures containing LLC-CM was shown by the loss of NS activity when CD34⁺ cells were depleted. The stimulation of CD34⁺ NS cells by LLC-CM was attributed to tumor production of GM-CSF, since neutralization of GM-CSF within the LLC-CM reduced its capacity to increase CD34⁺ cell levels. Studies also showed that the induction of CD34⁺ NS cells by LLC-CM and GM-CSF could be overcome by including in the cultures an inducer of myeloid differentiation, 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃]. These results demonstrate that the mechanism by which the LLC-LN7 tumors stimulate increased levels of CD34⁺ NS cells from normal bone marrow is by their production of GM-CSF and that this can be blocked with the myeloid differentiation inducer 1,25(OH)₂D₃. Received: 8 December 1997 / Accepted: 27 February 1998     

High Physiological Concentrations of Progesterone Reverse Estradiol-Mediated Changes in Differentiation and Functions of Bone Marrow Derived Dendritic Cells

Female sex steroids, estradiol (E2) and progesterone (P4), play a key role in regulating immune responses in women, including dendritic cell (DC) development, and functions. Although the two hormones co-occur in the body of women throughout the reproductive years, no studies have explored their complex combinatorial effects on DCs, given their ability to regulate each other’s actions. We examined murine bone marrow derived dendritic cells (BMDC) differentiation and functions, in the presence of a wide range of physiological concentrations of each hormone, as well as the combination of the two hormones. E2 (10⁻¹² to 10^-8M) enhanced the differentiation of CD11b⁺CD11c⁺ DCs from BM precursor cells, and promoted the expression of CD40 and MHC Class-II, in a dose-dependent manner. In contrast, P4 (10⁻⁹ to 10^-5M) inhibited DC differentiation, but only at the highest concentrations. These effects on BMDCs were observed both in the presence or absence of LPS. When both hormones were combined, higher concentrations of P4, at levels seen in pregnancy (10^-6M) reversed the E2 effects, regardless of the concentration of E2, especially in the absence of LPS. Functionally, antigen uptake was decreased and pro-inflammatory cytokines, IL-12, IL-1 and IL-6 production by CD11b⁺CD11c⁺ DCs, was increased in the presence of E2 and these effects were reversed by high concentrations of P4. Our results demonstrate the distinct effects of E2 and P4 on differentiation and functions of bone marrow myeloid DCs. The dominating effect of higher physiological concentrations of P4 provides insight into how DC functions could be modulated during pregnancy.     

Isolation, growth and identification of colony-forming cells with erythroid, myeloid, dendritic cell and NK-cell potential from human fetal liver

The study of hematopoietic stem cells (HSCs) and the process by which they differentiate into committed progenitors has been hampered by the lack of in vitro clonal assays that can support erythroid, myeloid and lymphoid differentiation. We describe a method for the isolation from human fetal liver of highly purified candidate HSCs and progenitors based on the phenotypes CD38⁻CD34⁺⁺ and CD38⁺CD34⁺⁺, respectively. We also describe a method for the growth of colony-forming cells (CFCs) from these cell populations, under defined culture conditions, that supports the differentiation of erythroid, CD14/CD15⁺ myeloid, CD1a⁺ dendritic cell and CD56⁺ NK cell lineages. Flow cytometric analyses of individual colonies demonstrate that CFCs with erythroid, myeloid and lymphoid potential are distributed among both the CD38⁻ and CD38⁺ populations of CD34⁺⁺ progenitors. Published: June 11, 2002.     

Identification of tissue-specific vasculogenic cells originating from murine uterus

Endometrium is a highly regenerative adult tissue that undergoes repeated degeneration and regeneration following menarche. Therefore, it is believed that endometrium contains stem and/or progenitor cells in order to compensate for the regeneration of tissue components. We report here that stem-like cells having vasculogenic potential are present in the uterus. Enzymatically extracted cells from murine uteri were characterized and fractionated into four subpopulations by flowcytometry; CD34⁺/45⁻ (Ut-34), CD34⁻/45⁻ (Ut-DN) and the remaining CD45⁺ cell fractions (CD34⁺/45⁺ and CD34⁻/45⁺ cells). The Ut-34 and Ut-DN fractions were mostly negative for putative endothelial cell (EC) markers, such as CD31, Flk-1, c-kit and VE-cadherin, although the Ut-DN fraction contained 2.8% CD31⁺ cells. Ut-DN cells were further divided into CD31⁺ and CD31⁻ fractions. Three cell populations were obtained from green fluorescence protein (GFP) transgenic mice and were transplanted into injured wild-type mouse skeletal muscle. At 4 weeks after cell transplantation, donor-derived vascular smooth muscle and ECs were observed in the injured recipient muscle. A similar trend was observed in the Ut-34 group, but differentiation into vascular smooth muscle was predominant. In contrast, the Ut-DN/31⁺ cell-transplanted group showed preferential differentiation into vascular ECs, thus suggesting that they were relatively committed preexisting ECs. These characteristics were also seen in vitro, in clonal cell cultures. Interestingly, donor derived Ut-DN/31⁺, Ut-DN/31⁻ and Ut-34 cells could not be identified after bone marrow (BM) transplantation, thus confirming that they are not derived from BM. It therefore appeared that tissue-specific vasculogenic cells are present in the murine uterus and that they exhibit vascular formation, even in different tissue microenvironments.     

Reciprocal Regulation of Development of Neutrophil-Dendritic Cell Hybrids in Mice by IL-4 and Interferon-Gamma

Neutrophils contribute to innate host immunity by functioning as professional phagocytes, whereas dendritic cells (DCs) are prototypic antigen presenting cells (APCs) responsible for the induction of adaptive immune responses. We have demonstrated recently that neutrophils trans-differentiate into a unique population, termed “neutrophil-DC hybrids,” expressing surface markers of both neutrophils and DCs and exhibiting dual functionality of both phagocytes and APCs. Although the hybrid cells emerged in significant numbers in murine bone marrow (BM) culture in the presence of GM-CSF, mechanisms regulating their development remained mostly unknown. In this study, we tested a total of 61 cytokines for their potentials to regulate neutrophil-DC hybrid formation using a newly developed BM micro-culture system combined with semi-automated FACS analysis. Several cytokines including GM-CSF were found to promote the generation of neutrophil-DC hybrids defined by the phenotype of CD11c⁺/MHC II⁺/Ly6G⁺. When tested in the presence of GM-CSF, hybrid cell development was enhanced by IL-4 and suppressed by interferon-γ (IFNγ) in dose-dependent fashions. We next determined in vivo impacts of IL-4 and IFNγ on the development of neutrophil-DC hybrids in thioglycollate-induced peritonitis lesions. Intraperitoneal administrations of IL-4/anti-IL-4 antibody complex (IL-4C) significantly increased the number of hybrids recovered from the lesions. By contract, recovery of hybrids was reduced by recombinant IFNγ. With regard to function, those hybrid cells recovered from IL-4C-treated mice and IFNγ-treated mice showed potent abilities to capture E.coli. These observations imply that emergence of neutrophil-DC hybrids in inflammatory sites is tightly regulated by local cytokine milieus.     

Immunotherapy with dendritic cells pulsed with tumor-derived gp96 against murine lung cancer is effective through immune response of CD8+ cytotoxic T lymphocytes and natural killer cells

Background and purpose Immunization with heat shock proteins, gp96, elicits specific protective immunity against parent tumors. However, it is marginally effective as a therapeutic tool against established tumors. In the present study, we evaluated the efficacy and mechanism of immunotherapy with bone marrow-derived dendritic cells (DCs) pulsed with tumor-derived gp96 against murine lung cancer. Methods Mice were transplanted subcutaneously with ovalbumin (OVA)-transfected Lewis Lung Cancer (LLC-OVA) cells and immunized with gp96 derived from LLC-OVA, DCs, or DCs pulsed with gp96 derived from LLC-OVA. Results The antitumor effect was significantly enhanced in the mice immunized with DCs pulsed with gp96 derived from LLC-OVA, compared to mice immunized with gp96 or DCs (P < 0.05). The antitumor effect was significantly dependent on natural killer (NK) cells and CD8⁺ cells and partially dependent on CD4⁺ cells. Analysis by laser confocal microscopy demonstrated that gp96 was shown on the cell surface at 15 min, and after 30 min internalized in the endosomes and not in the endoplasmic reticulum or lysosomes. OVA-specific⁺ CD8⁺ cells were more readily recruited into the draining lymph nodes and higher CD8⁺ cytotoxic T cell activity against LLC-OVA was observed in splenocytes from mice immunized with DCs pulsed with gp96 derived from LLC-OVA. Re-challenge of the surviving mice with LLC-OVA tumors after the initial tumor inoculation showed dramatic retardation in tumor growth. Conclusion In conclusion, immunotherapy of DCs pulsed with tumor-derived gp96 against murine lung cancer is effective through immune response of CD8⁺ cytotoxic T lymphocytes and NK cells.     

Differences amid bone marrow and cord blood hematopoietic stem/progenitor cell division kinetics

Human hematopoietic stem/progenitor cells (HSC) isolated based upon specific patterns of CD34 and CD38 expression, despite phenotypically identical, were found to be functionally heterogeneous, raising the possibility that reversible expression of these antigens may occur during cellular activation and/or proliferation. In these studies, we combined PKH67 tracking with CD34/CD38 immunostaining to compare cell division kinetics between human bone marrow (BM) and cord blood (CB)‐derived HSC expanded in a serum‐free/stromal‐based system for 14 days (d), and correlated CD34 and CD38 expression with the cell divisional history. CB cells began dividing 24 h earlier than BM cells, and significantly higher numbers underwent mitosis during the time in culture. By d10, over 55% of the CB‐cells reached the ninth generation, whereas BM‐cells were mostly distributed between the fifth and seventh generation. By d14, all CB cells had undergone multiple cell divisions, while 0.7–3.8% of BM CD34⁺ cells remained quiescent. Furthermore, the percentage of BM cells expressing CD34 decreased from 60.8 ± 6.3% to 30.6 ± 6.7% prior to initiating division, suggesting that downmodulation of this antigen occurred before commencement of proliferation. Moreover, with BM, all primitive CD34⁺CD38^? cells present at the end of culture arose from proliferating CD34⁺CD38⁺ cells that downregulated CD38 expression, while in CB, a CD34⁺CD38^? population was maintained throughout culture. These studies show that BM and CB cells differ significantly in cell division kinetics and expression of CD34 and CD38, and that the inherent modulation of these antigens during ex vivo expansion may lead to erroneous quantification of the stem cell content of the expanded graft. J. Cell. Physiol. 220: 102–111, 2009. © 2009 Wiley‐Liss, Inc.     

Vitamin A deficiency alters splenic dendritic cell subsets and increases CD8Gr-1 memory T lymphocytes in C57BL/6J mice

Vitamin A-deficient populations have impaired T cell-dependent antibody responses. Dendritic cells (DCs) are the most proficient antigen-presenting cells to naïve T cells. In the mouse, CD11b⁺ myeloid DCs stimulate T helper (Th) 2 antibody immune responses, while CD8α⁺ lymphoid DCs stimulate Th1 cell-mediated immune responses. Therefore, we hypothesized that vitamin A-deficient animals would have decreased numbers of myeloid DCs and unaffected numbers of lymphoid DCs. We performed dietary depletion of vitamin A in C57BL/6 J male and female mice and used multicolor flow cytometry to quantify immune cell populations of the spleen, with particular focus on DC subpopulations. We show that vitamin A-depleted animals have increased polymorphonuclear neutrophils, lymphoid DCs, and memory CD8⁺ T cells and decreased CD4⁺ T lymphocytes. Therefore, vitamin A deficiency alters splenic DC subpopulations, which may contribute to skewed immune responses of vitamin A-deficient populations.     

Age-related changes in the distribution and frequency of myeloid and T cell populations in the small intestine of calves

Mucosal dendritic cells (DCs) play a key role in discriminating between dietary antigens, commensal microflora and pathogens but little is known regarding age-related changes in mucosal DC populations. We analyzed lymphoid and myeloid populations within the epithelium and lamina propria (LP) of the ileum and jejunum of weaned calves (6 months old) and compared their frequency and distribution with newborn calves (3–5 weeks old). CD4, CD8 and γδ TcR T cells and CD11c^HiMHC Class II⁺ myeloid cell frequency were significantly different when comparing ileum and jejunum of weaned calves. In particular, the number of CD8 and γδ TcR T cells, and CD11c^HiCD14⁺ macrophages was significantly greater in the ileum but CD11c⁺ and CD11b⁺ myeloid cell distribution was similar throughout the mucosal epithelium of the small intestine. Furthermore, significant age-related changes were apparent when comparing the frequency and abundance of mucosal leukocyte subpopulations in newborn and weaned calves. Total mucosal leukocytes (CD45⁺) increased significantly with age in both ileum and jejunum and much of this increase was attributed to mucosal T cells (CD3⁺). In particular, CD4 T cells and NK cells increased significantly in the jejunum and CD8, and γδ TcR T cells increased significantly with age throughout the small intestine. In contrast, CD11c^HiMHC Class II⁺ myeloid cells remained numerically unchanged with age but DCs (CD13⁺, CD26⁺, CD205⁺) were enriched and macrophages (CD14⁺, CD172a⁺) were depleted in older animals. Therefore, regional differences between ileal and jejunal mucosal leukocytes changed with age and there was also a marked age-dependent change in the composition of mucosal myeloid cells. These observations have significant implications for host responses to both pathogens and commensal microflora.     

人树突状细胞的大量培养及鉴定

摘要 目的：探讨人树突状细胞体外大量培养及鉴定方法。方法：采用免疫磁珠法分离纯化CD34⁺干细胞;采用含有TPO、SCF、Flt3L和IL-3的扩增培养基培养1周,以及含有SCF、Flt3L、GM-CSF和IL-4的分化培养基培养2-3周,获得CD34⁺细胞来源树突状细胞。采用普通光学显微镜观察细胞形态,牛鲍氏血细胞计数板进行细胞计数,荧光抗体标记、流式细胞仪检测细胞纯度和细胞表面共刺激分子的表达情况。结果：以含有TPO、SCF、Flt3L和IL-3的培养基扩展培养一周,及含有SCF、Flt3L、GM-CSF和IL-4的培养基诱导分化3周,可获得大量悬浮细胞;细胞数目扩增倍数约达50倍;普通光学显微镜下可见悬浮细胞有明显的树突状凸起;流式细胞术检测结果显示悬浮细胞中CD141和CD11c双阳性细胞（等同于单核细胞来源树突状细胞）比例达30%,此群细胞高表达HLA-DR和CD209,低表达共刺激分子CD80和CD86;细胞寿命较短,40天时培养体系中悬浮细胞和CD34⁺细胞来源树突状细胞数目急剧减少。结论：采用多细胞因子联合刺激可获得大量的树突状细胞,为树突状细胞的特性及功能学研究奠定了基础。     

Cyclophosphamide induces bone marrow to yield higher numbers of precursor dendritic cells in vitro capable of functional antigen presentation to T cells in vivo

We have shown recently that cyclophosphamide (CTX) treatment induced a marked increase in the numbers of immature dendritic cells (DCs) in blood, coinciding with enhanced antigen-specific responses of the adoptively transferred CD8⁺ T cells. Because this DC expansion was preceded by DC proliferation in bone marrow (BM), we tested whether BM post CTX treatment can generate higher numbers of functional DCs. BM was harvested three days after treatment of C57BL/6 mice with PBS or CTX and cultured with GM-CSF/IL-4 in vitro. Compared with control, BM from CTX-treated mice showed faster generation and yielded higher numbers of DCs with superior activation in response to toll-like receptor (TLR) agonists. Vaccination with peptide-pulsed DCs generated from BM from CTX-treated mice induced comparable adjuvant effects to those induced by control DCs. Taken together, post CTX BM harbors higher numbers of DC precursors capable of differentiating into functional DCs, which be targeted to create host microenvironment riches in activated DCs upon treatment with TLR agonists.     

Granulocyte-macrophage colony-stimulating factor increases the proportion of circulating dendritic cells after autologous but not after allogeneic hematopoietic stem cell transplantation

Background aimsGranulocyte–macrophage (GM) colony-stimulating factor (CSF) has been used as an adjuvant in cancer immunotherapy. We tested the hypothesis that GM-CSF (Leukine^®; sargramostim) improves immune reconstitution after hematopoietic stem cell transplantation (HSCT) based on our prior in vitro work that demonstrated the pro-inflammatory effects of GM-CSF on dendritic cells (DC).MethodsGM-CSF was administered to donors, along with standard granulocyte (G) CSF, during stem cell mobilization, and to recipients from the day prior to transplant until engraftment. Eighteen patients consented to the GM-CSF⁺ protocol and were compared with 17 matched controls undergoing HSCT during the same time period (GM-CSF^?).ResultsNumbers of white blood cells (WBC) and CD34⁺ stem cells in the graft were comparable to controls. Surprisingly, contrary to our hypothesis, the allogeneic donor graft had significantly decreased numbers of CD3⁺ T cells and their subsets (CD4⁺, CD4⁺ CD45RA⁺, CD4⁺ CD45RO⁺, CD8⁺ and CD8⁺ CD45RO⁺), DC (both myeloid and plasmacytoid) and natural killer (NK) cells (CD16⁺ CD56⁺). In the GM-CSF arm, following allogeneic transplantation, the levels of DC, T cells and NK cells did not increase with treatment. Conversely, autologous transplant patients receiving GM-CSF had a higher proportion of DC at the time of engraftment.ConclusionsThese findings demonstrate that administration of GM-CSF improves DC reconstitution after autologous rather than allogeneic HSCT.     

Directed differentiation of human embryonic stem cells into functional dendritic cells through the myeloid pathway

We have established a system for directed differentiation of human embryonic stem (hES) cells into myeloid dendritic cells (DCs). As a first step, we induced hemopoietic differentiation by coculture of hES cells with OP9 stromal cells, and then, expanded myeloid cells with GM-CSF using a feeder-free culture system. Myeloid cells had a CD4+CD11b+CD11c+CD16+CD123(low)HLA-DR- phenotype, expressed myeloperoxidase, and included a population of M-CSFR+ monocyte-lineage committed cells. Further culture of myeloid cells in serum-free medium with GM-CSF and IL-4 generated cells that had typical dendritic morphology; expressed high levels of MHC class I and II molecules, CD1a, CD11c, CD80, CD86, DC-SIGN, and CD40; and were capable of Ag processing, triggering naive T cells in MLR, and presenting Ags to specific T cell clones through the MHC class I pathway. Incubation of DCs with A23187 calcium ionophore for 48 h induced an expression of mature DC markers CD83 and fascin. The combination of GM-CSF with IL-4 provided the best conditions for DC differentiation. DCs obtained with GM-CSF and TNF-alpha coexpressed a high level of CD14, and had low stimulatory capacity in MLR. These data clearly demonstrate that hES cells can be used as a novel and unique source of hemopoietic and DC precursors as well as DCs at different stages of maturation to address essential questions of DC development and biology. In addition, because ES cells can be expanded without limit, they can be seen as a potential scalable source of cells for DC vaccines or DC-mediated induction of immune tolerance.     

Flt3-ligand, IL-4, GM-CSF, and adherence-mediated isolation of murine lung dendritic cells: assessment of isolation technique on phenotype and function

Lung dendritic cells (DCs) are difficult to study due to their limited quantities and the complexities required for isolation. Although many procedures have been used to overcome this challenge, the effects of isolation techniques on lung DCs have not been reported. The current study shows that freshly isolated DCs (CD11c+) have limited ability to induce proliferation in allogeneic T cells, and are immature as indicated by low cell surface expression of costimulatory molecules compared with liver or splenic DCs. DCs isolated after overnight culture or from mice treated with Flt3L are phenotypically mature and potent stimulators of allogeneic T cells. DCs could not be propagated from lung mononuclear cells in response to IL-4 and GM-CSF. Contrary to data reported for nonpulmonary DCs, expression of CCR6 was decreased on mature lung DCs, and only a subset of mature DCs expressed higher levels of CCR7. Absence of CD8alpha expression indicates that freshly isolated DCs are myeloid-type, whereas mature DCs induced by overnight culture are both "lymphoid" (CD8alpha+) and "myeloid" (CD8alpha-). DCs from mice genetically deficient in CD8alpha expression were strong simulators of allogeneic T cells which was consistent with data showing that CD8alpha- DCs from CD8alpha-sufficient mice are better APCs compared with CD8alpha+ DCs from the same mice. These data show that freshly isolated lung DCs are phenotypically and functionally distinct, and that the isolation technique alters the biology of these cells. Therefore, lung DC phenotype and function must be interpreted relative to the technique used for isolation.